Vector borne flaviviruses, which belong to the Family Flaviviridae, genus Flavivirus, comprise some of the most important emerging and re-emerging viral pathogens. The tick borne flaviviruses (TBFV) include tick borne encephalitis virus (TBEV), Omsk hemorrhagic fever virus, Kyasanur forest disease virus, Alkhurma hemorrhagic fever virus, Powassan/deer tick virus (POWV/DTV) and Langat virus (LGTV). TBFV are generally transmitted to humans by ixodid ticks, and cause a spectrum of disease ranging from mild febrile illness to encephalitis, meningitis or hemorrhagic fevers. In the US, the cases of all tick-borne infections are increasing dramatically. POWV/DTV are the only autochthonous TBFV and case reports of serious illness and death are also on the increase. Importantly, POWV/DTV is transmitted by Ixodes scapularis ticks, the same vector that transmits the agent of Lyme disease, Borrelia burgdorferi, as well as several other emerging agents. Thus, a comprehensive evaluation of various tick-borne infections is needed to fully unravel the biology of the TBFV. The mosquito borne flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DEN) and yellow fever virus (YFV). The dramatic and ongoing pandemic attributed to the MBFV Zika virus is important because of its capacity to cause a severe congenital Zika disease as well an ever increasing spectrum of neurological syndromes in adults. Our current investigations are focused on the TBFV, but studying the biology of TBFV will illuminate the biology of other vector borne viruses. The research in our laboratory employs virology, immunology, entomology, advanced imaging techniques, genomics, cell biology, molecular biology, and vector biology. We study LGTV, a naturally attenuated member of the TBFV that can be safely studied at Biosafety Level-2 (BSL-2) making it an excellent model to gain insight into the TBFV. However, we are extending focus on the virulent autochthonous BSL-3 POWV/DTV. Neither of these 2 agents are Select Agents which greatly facilitates research studies. In addition, we have also continued to study the BSL2 MBFV, Zika virus. With the persistent emergence of Zika in the western hemisphere, similar avenues of inquiry are being explored for that vexatious pathogen. TBFV infection in ex vivo cultures of I. scapularis organs. Infection in ticks is patently a critical feature of TBFV biology, but it is woefully understudied. In the past year, we expanded our work using the ex vivo system in which salivary glands are successfully cultured and infected with TBFV and demonstrated that SG cultures prepared from fed ticks also supported TBFV replication. Earlier studies in tick cell culture showed that the gene vanin (VNN) played a role in TBFV infection. It is known that transmission of TBFV via tick bite can occur in less than 15 minutes. Our EM studies of POWV infected SG cultures revealed the presence of virus particles in cells of granular acini, the type of acini through to produce most salivary gland components. This finding likely explains how virus can be transmitted to so quickly once feeding begins. In addition, confocal microscopy of SG cultures infected with a GFP-expressing LGTV revealed that not all granular acini support infection. This finding suggested that virus may target specifc cell types in SG. Finally, when VNN expression in SG cultures was knocked down suing dsRNA, the replication of POWV in these cultures was also reduced. Thus, SG cultures likely provide a convenient and controlled system in which to look for genes associated with POWV replication and to assay antiviral countermeasures. Molecular biology and molecular pathogenesis of VBFV infection. The role of persistent infection in natural life cycle of VBFV is not well characterized, but may be responsible for prolonged debilitating sequelae observed in survivors of acute infection. The recent findings that ZIKV can persist in human semen for more than a year suggested that testicular cells might be targets for persistent ZIKV infection. In the past year we infected several testicular cell lines with several isolates of ZIKV. Persistent infection could be initiated but was of limited duration. In studies with intramural collaborators, we published evidence that infection of the testis occurs by a hematogenous route but that the epididymis can also occur by an excurrent testicular mechanism.